Unlike B and T cells, NK cells do not express antigen-specific receptors, yet they can eliminate virus-infected cells and cancer cells without harming normal cells. An important component that provides specificity in target cell recognition is inhibition of NK cells by inhibitory receptors that recognize surface molecules called major histocompatibility complex (MHC) class I. MHC-specific recognition by inhibitory receptors on NK cells prevents killing of normal healthy cells. The major goal of this project is to elucidate the mechanism by which inhibitory receptors block NK cell activation. Target cell killing by NK cells, which is called natural cytotoxicity, is achieved by polarized release of perforin and granzymes from lytic granules at the NKtarget cell immunological synapse. Signal transduction pathways that result in granule polarization and degranulation are uncoupled in NK cells. Therefore, we were interested in testing whether polarization and degranulation would have the same degree of sensitivity to inhibitory receptors. Expression of human MHC class I molecules, either HLA-C or HLA-E, on a class I-negative cell line blocked granule polarization, degranulation, and CD16-dependent cytokine secretion by NK cell clones that expressed inhibitory receptors of matching HLA specificity. To test inhibition of signals for polarization and degranulation separately, Drosophila S2 cells expressing ICAM-1 with either HLA-C or HLA-E were used. ICAM-1 is a ligand for the beta2 integrin LFA-1, which signals for granule polarization in NK cells. CD16-dependent degranulation and cytokine secretion were not fully inhibited, suggesting that other receptorligand interactions, which occur with human 721.221 cells, contribute to inhibition. In contrast, HLA-C or HLA-E on S2 cells co-expressing ICAM-1 were sufficient to block granule polarization induced by LFA-1, even during concomitant CD16-dependent degranulation. Therefore, granule polarization, rather than degranulation, is the preferred target of inhibitory receptors in NK cells. The main conclusion is that inhibitory receptors are better equipped to stop granule polarization than to block GrzB and chemokine release. Persistent degranulation during inhibition of polarization in IL-2-activated NK cells suggests that it may occur in high inflammatory conditions. Prevention of NK cell cytotoxicity would be better achieved through inhibition of degranulation rather than polarization. However, the possibility of releasing the block in degranulation while maintaining inhibition of polarization endows NK cells with the potential to provide bystander killing while still refraining from direct attacks on MHC class I-positive cells. Control of natural cytotoxicity by MHC class I-specific inhibitory receptors involves recruitment of the tyrosine phosphatase SHP-1, which dephosphorylates the guanine exchange factor Vav. As Vav is essential for proper actin remodeling and synapse formation, Vav inactivation through dephosphorylation provides an efficient way to block NK cell cytotoxicity. However, we have shown recently that the inhibitory signaling pathway is more complex and involves a second component, which relies on a tyrosine phosphorylation step. The small adapter Crk is phosphorylated during inhibition by MHC class I-specific receptors. Crk phosphorylation results in its dissociation from actin cytoskeleton-associated signaling complexes. Natural killer (NK) cell inhibitory receptors recruit tyrosine phosphatases to prevent activation, induce phosphorylation and dissociation of the small adaptor Crk from cytoskeleton scaffold complexes, and maintain NK cells in a state of responsiveness to subsequent activation events. How Crk contributes to inhibition is unknown. We imaged primary NK cells over lipid bilayers carrying IgG1 Fc to stimulate CD16, and human leukocyte antigen (HLA)-E to inhibit through receptor CD94-NKG2A. HLA-E alone induced Crk phosphorylation in NKG2A+ NK cells. At activating synapses with Fc alone, Crk was required for the movement of Fc microclusters and their ability to trigger activation signals. At inhibitory synapses, HLA-E promoted central accumulation of both Fc and phosphorylated Crk, and blocked the Fc-induced buildup of F-actin. We propose a unified model for inhibitory receptor function: Crk phosphorylation prevents essential Crk-dependent activation signals and blocks F-actin network formation, thereby reducing constraints on subsequent engagement of activation receptors.